The toxicity of sterols to cells is a significant component of several disease syndromes ranging from Alzheimer's disease to Atherosclerosis. We hypothesize that a pivotal component of obviating this threat is the removal of the offending metabolite to non-toxic environments, for neutralization by esterification or efflux to extracellular acceptors. The endoplasmic reticulum is a critical organelle with regard to sterol homeostasis. Sterol levels in this organelle are maintained at very low levels, relative to the plasma membrane, in part by anteriograde transport. We have identified an evolutionary conserved component of this transport, Arvlp that directs the egress of sterol from the endoplasmic reticulum to the plasma membrane. Furthermore, one consequence of loss of Arvlp is induction of the unfolded protein response. The association of this pathway with membrane related changes is unprecedented and will likely impact pathologies that arise from protein misfolding (e.g. prion disorders). Our studies will utilize genetic approaches to understand the mechanisms and consequences of ARVl-mediated sterol transport. In this revised proposal, we will continue to exploit genetic and cell biology approaches in yeast, and expand these approaches to understanding the role of Arvl in higher cells and multicellular organisms. Our specific aims and hypotheses are: 1. To define the mechanisms by which Arvlp mediates sterol transport in yeast. We hypothesize that the structure of ARV1 (Zn-binding motif and transmembrane nature) is required for sterol transport and that these domains mediate ligand (lipid and protein) binding. 2. To identify and characterize new components of sterol homeostasis. We hypothesize that ARV1 represents a decision point in terms of responses to sterol overloading of a cell. Factors that interact with ARV1 will be critical to sterol homeostasis and cell survival. One example of such a pathway is the Unfolded Protein response (UPR) which is induced by loss of ARVl. We will identify and analyze these pathways using genetic and biochemical approaches. 3. To define the role of Arvlp in mammalian cells. We hypothesize that the role of ARVl in human cells is also to transport sterol to the plasma membrane. We will test the ramifications of overexpression or knockdown of human ARVl in various tissue culture cell models. Lipid efflux to extracellular acceptors will be studied in detail.